Infective endocarditis affects about 22,000 people per year in the U.S. and kills over 2,000. This disease is caused primarily by viridans streptococci, and among viridans streptococci, primarily by Streptococcus sanguis. Previous studies concerning the virulence of streptococci for endocarditis have centered upon the examination of suspected virulence factors. The proposed research takes an entirely different approach, that of directly identifying new virulence factors. This will be done by adapting the technique of signature-tagged mutagenesis (STM) for use with S. sanguis. This approach requires no prior assumptions concerning the importance of any particular gene or activity in disease causation. Instead, it relies on the infection process to identify the bacterial genes needed for disease and their relative contributions. An S. sanguis strain that is also the subject of a separate ongoing genome sequencing project will be randomly mutagenized by STM using an in vitro transposition approach. The resulting mutants will be pooled and inoculated into rats serving as models for endocarditis. The STM approach will allow for the identification of mutants within the pool possessing increased or decreased virulence. The apparent altered virulence in these mutants will be verified by in vivo competition assays. Further characterization of mutants will include DNA sequence analysis to identify the mutated genes, further genetic manipulation to verify the contribution of the mutated genes, in vivo infectivity assays, and in vitro functional assays. It is anticipated that the characterization of mutants with altered virulence will result in a better understanding of the pathogenesis of endocarditis and will identify promising novel targets for vaccine or therapeutic development.